Project Summary Over the past two decades, disseminated Mycobacterium tuberculosis (MTB) has been increasingly recognized as the leading cause of fatal bloodstream infection in HIV-infected patients with sepsis in high- burden tuberculosis (TB)/HIV settings worldwide. In these settings, estimated prevalence of MTB bacteremia in HIV-infected adults presenting with sepsis is as high as 40% with mortality approaching 50%. Despite high prevalence and mortality, timely and accurate diagnosis of MTB bacteremia and concomitant TB drug resistance remains challenging and is often diagnosed post-mortem. Novel diagnostic strategies are needed to expedite identification and treatment of critically ill patients with MTB bacteremia and other severe bloodstream infections in high-burden TB/HIV settings. The overall goal of this study is to enhance rapid molecular detection of MTB and other high-impact bacterial pathogens among HIV-infected adults with sepsis through use of an innovative, highly sensitive next-generation sequencing approach. We propose to utilize a novel positive- selection nucleic acid enrichment and high-throughput sequencing platform (BacCapSeq) to detect MTB bacteremia and drug-resistant organisms, optimized at the bench and trialed in a nested case control study of HIV-associated sepsis in Uganda. Specifically, we will evaluate the performance of BacCapSeq and emerging, near-point-of-care diagnostics (Xpert MTB/RIF Ultra) for the detection and characterization of MTB bacteremia and MTB drug resistance using biobanked samples from a prospective cohort of HIV-infected adults with sepsis in Uganda (Aim 1); and identify and characterize high-priority, antibiotic-resistant bacterial pathogens associated with sepsis in HIV-infected patients without MTB infection in Uganda (Aim 2). At the conclusion of this work we will have addressed critical diagnostic gaps for severe HIV/TB disease while providing high- resolution genomic characterization of pathogens and antimicrobial resistance associated with sepsis, a frequent pathway to death for HIV-infected adolescents and adults worldwide.